Electrophoretic apparatus employing a collecting belt moving in contact with a gel

ABSTRACT

Apparatus for performing electrophoresis in an electrical field in which molecules or charged particles are moved through a porous substance such as a gel and a collecting tape is transported in contact with the gel to collect charged particles passing through the gel.

OTHER APPLICATIONS

This application is a divisional application based on my copendingapplication Ser. No. 273,116, filed June 12, 1981.

FIELD OF INVENTION

The invention relates to apparatus for performing electrophoresis in anelectrical field, whereby molecules or particles are moved in a porousmaterial, for example a gel, and which are, for example, made detachableby optical means.

BACKGROUND

Electrophoresis, an electrokinetic phenomenon, is used mainly inanalytical and clinical chemistry for analyzing mixtures of substances,where one determines the different mobility of ions in an electricfield. In addition, electrophoresis in porous media--such aspolyacrylamide or agarose gels--has been developed to an importantanalytical and also preparative method in biochemistry and medicine,e.g., for separating complex mixtures of nucleic acids or proteins intosingle components. After the application of an electrical field,different molecules move different distances into the gel (whichincludes all the substances suitable for this purpose) dependent ontheir mobility and are, thereby, separated in space. By specificstaining of these molecules, their distribution in the mixture can bevisualized and analyzed. A disadvantage is that the gel has to beremoved from the electrophoresis apparatus and that it can be used onlyonce. To achieve a satisfactory separation of many components--forexample in the sequence analysis of DNA including up to a few hundredmolecules of different lengths--gels of up to one meter length arecurrently used; further, a very high voltage has to be applied to obtaina large voltage gradient, which allows a separation within reasonabletime.

In preparative electrophoresis, a stationary collection chamber isusually provided at the end of the gel, into which the molecules move.This chamber is emptied from time to time and is then filled again; inthis way, a stepwise separation of components of the mixture can beachieved.

SUMMARY OF INVENTION

In view of the above, it is an object of the invention to provide animproved apparatus for the electrophoresis so that the use of theapparatus is simplied, the length of the gel reduced, and the wholeprocedure performed automatically, if required. To solve this problem inaccordance with the invention is to provide at least one binding orcollecting surface which moves continuously or which stepwise passes byin the area to which the molecules migrate, for example at the end ofthe porous substance (e.g., the gel). A further characteristic of themethod of the invention is that the molecules are continuously collectedon the binding surface due to ion exchange or reactive chemical groups.

The invention furthermore provides a device wherein at least onetapelike binding surface moves across the porous substance (e.g., thegel) and which is constructed as a continuously moving or circulatingconveyer belt (hereafter termed as "the tape").

This conveyer belt may consist of several layers; the layer facing thegel is used for binding the molecules, while another layer supports thebinding surface. This allows, for example, the use of binding surfacesof minor mechanical strength which are supported by a plastic net movedby a driving unit.

Especially useful are binding surfaces consisting of paper, foils orsimilar material, which have a surface charge opposite that of the ionicmolecules, or binding surfaces with reactive chemical groups forcovalent or non-covalent binding of molecules.

In a rather short and solid porous phase, which is denoted herein as gel(such as polyacrylamide gel, agarose gel, paper, foil, or similarsubstances) to which a direct current is applied, the charged moleculesor particles move according to their mobility to the end of the gel andare bound there to the moving tape, that is at the charged surface(ion-exchange paper) or the reactive chemical groups thereof. Accordingto another characteristic of the invention, the tape can be movedthrough a fixation or staining bath so that the components becomevisible and can be recorded. It is also within the purpose of theinvention to elute and collect single components and to use conditionswhere any interaction between particles and tape is eliminated.

The width of an eluted component on the tape or conveyer-belt will besmaller the thinner the gel, so that, for analytical problems, gels asthin as possible should be used. However, according to a furthercharacteristic of the invention, components can be concentrated byforming an angle between the direction of the eluting and movingparticles and the direction of the moving tape. The binding surfacecollects the molecules first at the point where it touches the gel and,while it moves on, the particles are eluted and bound at the same point.This may be important for analytical purposes, but in particular isimportant for preparative purposes. On the other hand, it is possible todistribute a component over a large area by using a high tape velocity,thereby overcoming possible limitations in the binding capacity.

The use of a movable tape onto which particles or molecules are boundafter leaving the gel adds an additional dimension to electrophoreticseparations. The distance between components on the tape can be easilyvaried by changing the tape velocity. Furthermore, the relative positionof components on the tape can be varied by programming the tapevelocity, the voltage gradient, or the temperature.

Use of different materials for the tape, which may contain more than onelayer, is within the scope of the invention. Thus, detection methodssuch as staining, radioactivity, etc., are no longer bound to the gelmaterial, which results in higher flexibility.

An especially important feature of the invention is that a separation isachieved between electrophoresis and detection on the tape, allowing oneto activate reactive groups immediately before use, while not onlystaining or other analytical procedures, but also elution of boundmolecules or particles after electrophoresis can be done in acontinuous, automatic process in different baths or devices. The tapecan be washed after analysis or elution and used again, which allowsmaterial to be saved.

After all components of a mixture have left the gel, a new mixture canbe applied to it. Thus, considerable savings in material and preparationtime for gels are possible and the separations become more reproducible.Use of short gels, as mentioned before, also saves material and offersthe additional advantage that the time during which fast-movingmolecules remain in the gel is shortened, the diffusion minimized, andthe separation improved. The total voltage for achieving a certainvoltage gradient can be reduced for short gels, thus improving security,decreasing energy demand, and allowing for simple removal of thegenerated heat.

The molecules of a component can be geometrically concentrated in thecontact area between gel and tape to a zone narrower than the thicknessof the gel. This can result in higher analytical sensitivity and betterresolution for preparative separations. Of advantage is to position thegel in a vertical shaft with an opening facing the conveyer belt. Thelength of the opening can be chosen so that a number of parallelconveyer belts are possible, which may move at different speeds. It mayalso be of advantage to employ a triangular opening, so as to make itnarrower on one side of the tape or above the adjacent tapes; such aconfiguration offers new possibilities when a number of parallel tapesare employed. Furthermore, for the use of different gels in parallel, agel-bearing surface with a number of parallel grooves has proved to befavorable.

In practice, it has been shown that the method and device according tothe invention can be used for analytical, as well as for preparativeseparations, and thus considerably expand the field of applications.

BRIEF DESCRIPTION OF DRAWING

Further advantages, characteristics and details of the invention appearin the following description of preferred embodiments and in thedrawings in which:

FIG. 1 is a schematic cross-section of a device according to invention;

FIG. 2 is a top-view of an enlarged detail of another device of theinvention, alone line II--II in FIG. 1;

FIG. 3 is an enlarged cross-section of a detail of a device according tothe invention;

FIG. 4 is another detail of a device of the invention;

FIG. 5 is a different design of the device of the invention; and

FIG. 6 is a diagonal view of a detail of another design.

DETAILED DESCRIPTION

In FIG. 1, a buffer vessel 1 of an apparatus E for performingelectrophoresis in accordance with the invention incorporates at itsaxis A an electrode 2 which, together with a second electrode 3 at adistance h below, creates an electrical field (not shown). Within itsrange is a vertical shaft 4 of width b and height i for the gel G.

At both sides of the shaft, which ends at the bottom of the vessel 5,chambers 6 are provided for temperature control. In FIG. 1, the walls 7of the vessel 1 are parallel to the walls 8 of the shaft 4 and areconnected at their lower ends by the curved outer walls 9 of thechambers. A tape 11 is positioned below the opening 10 of the shaft 4,which moves over the roller 12 in a horizontal direction as shown by thearrow x below the opening 10 of the shaft 4. The tape 11 and the opening10 are summerged in the solution 13 contained in the vessel 14.

The tape 11 leaves the solution 13 across the guide pulley 15 and itsmovement is controlled by a driving shaft 16 and a pressure roller 17.

The ions leaving the gel G are bound on a filter layer 20 (e.g., filterpaper) of the tape 11 and leave the solution 13 horizontally. The fillerlayer 20 is carried by a supporting net 21.

While the width b of the shaft 4 is constant in the device E of FIG. 1,FIG. 2 shows an opening 10 of the shaft 4, the width b₁ of whichincreases toward one of the vessels, with several tapes 11a, 11b, and11c moving below the opening.

The single-layer tape 11 in FIG. 3 is below a shaft 4, the width ofwhich narrows toward the opening 10.

FIG. 4 shows a two-layer tape 11 at an angle w with respect to the gelG, first touching the gel at 23. The ions of the gel area at distance bfrom 23 arrive at tape 11 later, at a position that depends on theirvelocity v₁ and the tape velocity v₂.

In another example of a device according to the invention, the tape 11moves vertically (FIG. 5); it runs through a fixing or washing bath 24and touches the gel G, which rests on the tunnel 25, at a vertical area26. The mixture in this device E₁ is applied via 27.

FIG. 6 shows a top surface 28 of tunnel 25 with a number of grooves 29for using different gels G₁, G₂, G₃.

I claim:
 1. Apparatus for performing an electrophoretic operationcomprising first means for accommodating a porous substance, secondmeans for providing an electrical field traversing said poroussubstance, displacable tape collection means adjacent the poroussubstance, and drive means driving said tape collection means to providerelative movement between the tape collection means and the poroussubstance whereby to effect said electrophoretic operation on anelectrophoretically responsive substance inclusive of substances capableof migrating through the porous substance under the influence of saidelectrical field.
 2. Apparatus according to claim 1, wherein saidcollection means is in the form of a conveyer belt.
 3. Apparatusaccording to claim 2, including at least one cleaning bath to receivethe conveyer belt downstream of the contact between the porous substanceand the conveyer belt.
 4. Apparatus according to claim 2, wherein saidconveyer belt includes at least first and second layers, wherein thefirst layer faces the porous substance and serves to collect thesubstances migrating through the porous substance and the second layeracts as a support for the first layer.
 5. Apparatus according to claim4, wherein the supporting layer is a net.
 6. Apparatus according toclaim 4, wherein the first layer consists of a substance with a surfacecharge inverse to that of the migrating substances.
 7. Apparatusaccording to claim 4, wherein the first layer includes reactive chemicalgroups for the covalent or non-covalent binding of the migratingsubstances.
 8. Apparatus according to claim 4, wherein the conveyer beltincludes a portion positioned vertically to contact the poroussubstance.
 9. Apparatus according to claim 4, wherein said first meansis provided with a vertically oriented shaft having an opening above theconveyer belt.
 10. Apparatus according to claim 9, wherein said shafthas a cross-section narrowing toward said opening.
 11. Apparatusaccording to claim 9 wherein said opening has variable width. 12.Apparatus according to claim 11, wherein the opening has a triangularcross-section.
 13. Apparatus according to claim 4, wherein the migratingsubstance migrates in a determinable direction and the conveyer beltincludes a portion which moves at an angle with respect to the directionof migration.
 14. Apparatus according to claim 4, comprising means forcontrolling the velocity of the conveyer belt.
 15. Apparatus accordingto claim 14, including computer means coupled to said drive means tocontrol the drive means.
 16. Apparatus according to claim 4, includingat least one bath means in which to receive and submerge the conveyerbelt.
 17. Apparatus according to claim 16 wherein the second means ispositioned in said bath means.
 18. Apparatus according to claim 4wherein the conveyer belt is positioned to make tangential slidingcontact with the porous substance.
 19. Apparatus according to claim 18wherein said drive means comprises drive wheels drivingly engaging saidconveyor belt to advance the belt past the porous substance. 20.Apparatus according to claim 1 wherein the collection means is aconveyer belt including a plurality of adjacent tapes.
 21. Apparatusaccording to claim 1 including a temperature control means forcontrolling the temperature of the porous substance.
 22. Apparatusaccording to claim 1 comprising means having a support surface with aplurality of grooves each for receiving a respective the first poroussubstance.
 23. Apparatus according to claim 1 constituting said poroussubstance comprising a gel.